Mutant aspartokinase gene

ABSTRACT

Recombinant DNA replicable in microorganisms belonging to the genus Corynebacterium, which contains a DNA fragment coding for an aspartokinase α-subunit protein originating from a bacterium belonging to the genus Corynebacterium, in which synergistic feedback inhibition by L-lysine and L-threonine is substantially desensitized, and a DNA fragment coding for an aspartokinase β-subunit protein originating from a bacterium belonging to the genus Corynebacterium, in which synergistic feedback inhibition by L-lysine and L-threonine is substantially desensitized, is introduced into a microorganism belonging to the genus Corynebacterium. Thus a transformant having enhanced production and excretion speeds of L-lysine is obtained. The transformant is cultivated in an appropriate medium, and produced L-lysine is separated.

TECHNICAL FIELD

The present invention relates to a novel aspartokinase and a DNAfragment coding for the enzyme originating from a bacterium belonging tothe genus Corynebacterium used for fermentative production of amino acidand so on, and relates to recombinant DNA containing the DNA fragment.The present invention also relates to a bacterium belonging to the genusCorynebacterium harboring the recombinant DNA, and relates to a methodof producing L-lysine comprising cultivating the microorganism.

BACKGROUND ART

L-Lysine, which is used as a feed additive, is usually produced byfermentation by using an L-lysine-producing mutant strain belonging tocoryneform bacteria. A variety of L-lysine-producing bacteria are knownat present, which are those created by artificial mutation of coryneformbacteria. Such artificial mutant strains includes the followings:S-(2-aminoethyl)cysteine (hereinafter abbreviated as "AEC") resistantmutant strains; mutant strains which require amino acid such asL-homoserine for their growth (Japanese Patent Publication Nos. 48-28078and 56-6499); mutant strains which exhibit resistance to AEC and requireamino acids such as L-leucine, L-homoserine, L-proline, L-serine,L-arginine, L-alanine, and L-valine (U.S. Pat. Nos. 3,708,395 and3,825,472); L-lysine-producing mutant strains which exhibit resistanceto DL-α-amino-ε-caprolactam, α-amino-lauryllactam, aspartic acid-analog,sulfa drug, quinoid, and N-lauroylleucine; L-lysine-producing mutantstrains which exhibit resistance to inhibitors of oxyaloacetatedecarboxylase or respiratory system enzymes (Japanese Patent Laid-openNos. 50-53588, 50-31093, 52-102498, 53-9394, 53-86089, 55-9783, 55-9759,56-32995 and 56-39778, and Japanese Patent Publication Nos. 53-43591 and53-1833); L-lysine-producing mutant strains which require inositol oracetic acid (Japanese Patent Laid-open Nos. 55-9784 and 56-8692);L-lysine-producing mutant strains which exhibit sensitivity tofluoropyruvic acid or temperature not less than 34° C. (Japanese PatentLaid-open Nos. 55-9783 and 53-86090); and mutant strains belonging tothe genus Brevibacterium or Corynebacterium which exhibit resistance toethylene glycol and produce L-lysine (U.S. patent application Ser. No.333,455).

Escherichia coli transformed by using a recombinant vector is disclosedin the prior art. In this strain, amino acid production is enhanced (seeU.S. Pat. No. 4,278,765).

In relation to the genera Brevibacterium and Corynebacterium, there aredisclosed a vector plasmid which is autonomously replicable in bacterialcells and has a drug resistance marker gene (see U.S. patent applicationSer. No. 386,980), and a method for introducing genes into bacterialcells (Japanese Patent Laid-open No. 2-207791). There is disclosed apossibility to breed L-threonine- or L-isoleucine-producing bacteria byusing the techniques described above (see U.S. patent application Ser.Nos. 376,396 and 392,145). In relation to breeding of L-lysine-producingbacteria, a technique is known in which a gene relevant to L-lysinebiosynthesis is integrated into a vector plasmid to amplify it inbacterial cells (for example, Japanese Patent Laid-open No. 56-160997).However, no prior art is known in which a gene is specified foraspartokinase (hereinafter referred to as "AK"), a mutation point on theAK gene is elucidated so that feedback inhibition by L-lysine andL-threonine is substantially desensitized, and it is elucidated that themutation directly relates to productivity of L-lysine. Although a mutantAK gene is described in a few cases, the mutant AK gene could not beharbored as a stable plasmid (see Cremer, J. et al.; Applied andEnvironmental Microbiology, June 1991, pp. 1746-1752).

An object of the present invention is to make improvement to provideincreased production and secretion speeds of L-lysine by modifying AK asan important enzyme for lysine biosynthesis in microorganisms ofbacteria belonging to the genus Corynebacterium into those having aproperty of desensitization of feedback inhibition by L-lysine andL-threonine and feedback inhibition by L-lysine alone, and increasingtheir activities.

DISCLOSURE OF THE INVENTION

As a result of diligent studies, the present inventors have succeeded inobtaining mutant AK genes from a bacterium belonging to the genusCorynebacterium, and completed the present invention. Namely, thepresent invention lies in an aspartokinase α-subunit protein and a DNAfragment coding for the protein originating from a bacterium belongingto the genus Corynebacterium, in which synergistic feedback inhibitionby L-lysine and L-threonine is substantially desensitized, the proteinhaving an amino acid sequence defined in SEQ ID NO: 4 in SequenceListing, or a sequence in which a 279th Thr residue in the amino acidsequence defined in SEQ ID NO: 4 is changed to an amino acid residueother than Ala and other than acidic amino acids. Further, the presentinvention lies in an aspartokinase β-subunit protein and a DNA fragmentcoding for the protein originating from a bacterium belonging to thegenus Corynebacterium, in which synergistic feedback inhibition byL-lysine and L-threonine is substantially desensitized, the proteinhaving an amino acid sequence defined in SEQ ID NO: 6 in SequenceListing, or a sequence in which a 30th Thr residue in the amino acidsequence defined in SEQ ID NO: 6 is changed to an amino acid residueother than Ala and other than acidic amino acids.

In another aspect, the present invention lies in recombinant DNAcontaining the aforementioned DNA fragment and being replicable inmicroorganisms belonging to the genus Corynebacterium, and atransformant obtained by introducing the recombinant DNA into amicroorganism belonging to the genus Corynebacterium, wherein thespecific activity of aspartokinase is increased 2-20 times as comparedwith a parent strain, and synergistic feedback inhibition by L-lysineand L-threonine or feedback inhibition by L-lysine alone exerted on theactivity of aspartokinase is substantially desensitized.

In another aspect, the present invention lies in a method of producingL-lysine comprising the steps of cultivating the aforementionedtransformant in an appropriate medium, and separating produced L-lysine.

The microorganisms belonging to the genus Corynebacterium referred to inthe present invention are a group of microorganisms as defined inBergey's Manual of Determinative Bacteriology, 8th ed., p. 599 (1974),which are aerobic gram-positive rods having no acid resistance and nospore-forming ability. The microorganisms belonging to the genusCorynebacterium referred to in the present invention include bacteriabelonging to the genus Brevibacterium having been hitherto classifiedinto the genus Brevibacterium but united as bacteria belonging to thegenus Corynebacterium at present, and include bacteria belonging to thegenus Brevibacterium closely relative to bacteria belonging to the genusCorynebacterium. Among the microorganisms belonging to the genusCorynebacterium (Brevibacterium) as described above, especially glutamicacid-producing bacteria belonging to the genus Corynebacterium(Brevibacterium) as mentioned below are most preferable in the presentinvention. Further, some bacteria belonging to the genus Microbacteriumare known to accumulate glutamic acid, which can be also used in thepresent invention.

Examples of wild type strains of glutamic acid-producing bacteriabelonging to the genus Corynebacterium (Brevibacterium) include thefollowings.

    ______________________________________                                        Corynebacterium acetoacidophilum                                                                     ATCC 13870                                             Corynebacterium acetoglutamicum                                                                      ATCC 15806                                             Corynebacterium callunae                                                                             ATCC 15991                                             Corynebacterium glutamicum                                                                           ATCC 13032                                                                    ATCC 13060                                             (Brevibacterium divaricatum)                                                                         ATCC 14020                                             (Brevibacterium lactofermentum)                                                                      ATCC 13869                                             Corynebacterium lilium ATCC 15990                                             Corynebacterium melassecola                                                                          ATCC 17965                                             Brevibacterium saccharolyticum                                                                       ATCC 14066                                             Brevibacterium immariophilum                                                                         ATCC 14068                                             Brevibacterium roseum  ATCC 13825                                             Brevibacterium flavum  ATCC 13826                                             Brevibacterium thiogenitalis                                                                         ATCC 19240                                             Microbacterium ammoniaphilum                                                                         ATCC 15354                                             ______________________________________                                    

The glutamic acid-producing bacteria belonging to the genusCorynebacterium (Brevibacterium) of the present invention also includemutant strains having glutamic acid productivity or having lost glutamicacid productivity, in addition to the wild type strains having glutamicacid productivity as described above.

When a wild type strain is used as a donor for the DNA fragmentcontaining the AK gene, a DNA fragment containing a wild type AK genecan be obtained. The DNA fragment containing the gene for AK in whichsynergistic feedback inhibition by L-lysine and L-threonine issubstantially desensitized can be obtained by using a mutant strain inwhich synergistic feedback inhibition on the AK activity by L-lysine andL-threonine is substantially desensitized. The mutant strain can beobtained, for example, from a group of cells subjected to an ordinarymutation treatment, ultraviolet light irradiation, or a treatment with amutating agent such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG). TheAK activity can be measured by using a method described in Miyajima, R.et al., The Journal of Biochemistry (1968), 63(2), 139-148.

As for the donor for the DNA fragment containing the AK gene,Corynebacterium glutamicum (Brevibacterium lactofermentum) wild typestrain ATCC 13869, and an L-lysine-producing bacterium AJ3463 (FERMP-1987) derived by a mutation treatment from the ATCC 13869 strain aremost preferable donors. The wild type AK gene, and the gene coding foraspartokinase in which synergistic feedback inhibition by L-lysine andL-threonine is substantially desensitized (hereinafter referred to as"mutant AK gene") are separated from chromosomal DNA of these bacteria,ligated with a vector autonomously replicable in bacteria belonging tothe genus Corynebacterium (Brevibacterium), and introduced into cells ofbacteria belonging to the genus Corynebacterium (Brevibacterium).

A method for isolating the AK gene is as follows. At first, achromosomal gene is extracted from a strain having the AK gene of abacterium belonging to the genus Corynebacterium (for example, a methodof H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963) can beused), and it is digested with a suitable restriction enzyme.Subsequently, it is ligated with a vector replicable in cells ofbacteria belonging to the genus Corynebacterium. An obtained recombinantvector is used to transform an AK-deficient mutant strain of amicroorganism belonging to the genus Corynebacterium. A bacterial stainconsequently harboring the AK-producing activity is isolated, from whichthe AK gene can be separated. A method for deriving the AK-deficientmutant strain is similar for practice to a method for deriving themutant strain which provides substantially desensitized synergisticfeedback inhibition on the AK activity by L-lysine and L-threoninedescribed above.

Upon digestion of the chromosomal gene, the degree of digestion can becontrolled by controlling the digestion reaction time and so on. Thus awide variety of restriction enzymes can be used.

Any vector replicable in cells of bacteria belonging to the genusCorynebacterium can be used as the vector for the present invention.Specifically, it is exemplified by the followings.

(1) pAM330: see Japanese Patent Laid-open No. 58-67699

(2) pHM1519: see Japanese Patent Laid-open No. 58-77895

(3) pAJ655: see Japanese Patent Laid-open No. 58-192900

(4) pAJ611: see the same

(5) pAJ1844: see the same

(6) pCGi: see Japanese Patent Laid-open No. 57-134500

(7) pCG2: see Japanese Patent Laid-open No. 58-35197

(8) pCG4: see Japanese Patent Laid-open No. 57-183799

(9) pCG11: see the same

The vector may be cleaved by digestion with a restriction enzyme whichdigests the DNA at one place, or by partial digestion with a restrictionenzyme which digests it at a plurality of places.

The vector is digested with a restriction enzyme used to digest thechromosomal gene, or ligated with oligonucleotides having nucleotidesequences complementary to both ends of the digested fragment ofchromosomal DNA and the digested vector respectively. Subsequently, itis subjected to a ligation reaction of the vector and the chromosomalDNA fragment.

Recombinant DNA constructed by ligating the chromosomal DNA with thevector thus obtained can be introduced into a recipient of bacteriabelonging to the genus Corynebacterium by using a method in whichpermeability of DNA is increased by treating recipient cells withcalcium chloride as reported for Escherichia coli K-12 (Mandel, M. andHiga, A., J. Mol. Biol., 53, 159 (1970)), or by using a method in whichintroduction is performed in a proliferating stage (so-called competentcells) so that cells can incorporate DNA as reported for Bacillussubtills (Duncan, C. H., Wilson, G. A. and Young, F. E., Gene, 1, 153(1977)). Alternatively, recombinant DNA can be introduced into a DNArecipient after converting DNA recipient cells into protoplasts orspheroplasts which easily incorporate recombinant DNA as known forBacillus subtills, actinomycetes, and yeasts (Chang, S. and Choen, S.N., Molec. Gen. Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. andHopwood, O. A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B. andFink, G. R., Proc. Natl. Acad. Sci. U.S.A., 75, 1929 (1978)).

In the protoplast method, a sufficiently high frequency can be obtainedeven by using the method used for Bacillus subtills described above. Itis of course possible to utilize a method in which DNA is incorporatedinto protoplasts of a bacterium belonging to the genus Corynebacteriumin the presence of polyethylene glycol or polyvinyl alcohol and divalentmetal ion as described in Japanese Patent Laid-open No. 57-183799. Anequivalent result is obtained even by using a method in whichincorporation of DNA is facilitated by addition of carboxymethylcellulose, dextran, Ficoll, Bruronik F68 (Serva) instead of polyethyleneglycol or polyvinyl alcohol.

Alternatively, the AK gene can be obtained by amplifying the AK gene byusing PCR (polymerase chain reaction; see White, T. J. et al., TrendsGenet., 5, 185 (1989)) from chromosomal DNA obtained as described above.DNA primers to be used for the amplification are those complementary toboth 3' ends of DNA double strands containing an entire or partialregion of the AK gene. When only a partial region of the AK gene isamplified, it is necessary to perform screening from a gene library byusing DNA fragments of the region as primers to amplify a DNA fragmentcontaining an entire region. When an entire region is amplified, a DNAfragment containing the AK gene can be recovered by excising anobjective band after subjecting the DNA fragment to agarose gelelectrophoresis.

For DNA primers, single strand DNA's of 23 mer and 21 mer havingsequences of 5'-TCGCGAAGTAGCACCTGTCACTT-3' (SEQ ID NO:15) and5'-ACGGAATTCAATCTTACGGCC-3' (SEQ ID NO:16) are most suitable to amplifya region of about 1,643 bp coding for the AK gene based on, for example,a sequence known for Corynebacterium glutamicum (see MolecularMicrobiology (1991), 5(5), 1197-1204; Mol. Gen. Genet. (1990), 224,317-324). The DNA can be synthesized in accordance with an ordinarymethod using a phosphoramidite method (see Tetrahedron Letters (1981),22, 1859) by using a DNA synthesizer Model 380B produced by AppliedBiosystems. The PCR can be performed by using DNA Thermal Cycler ModelPJ2000 produced by Takara Shuzo Co., Ltd., using Taq DNA polymerase inaccordance with a method designated by the supplier.

The amplified AK gene is ligated with the vector proliferative in cellsof bacteria belonging to the genus Corynebacterium as described above,and introduced into cells of bacteria belonging to the genusCorynebacterium by using the method as described above.

Hosts in which the obtained AK gene is introduced and amplified toproduce lysine include the wild type strains of glutamic acid-producingbacteria belonging to the genus Corynebacterium described above. Allbacteria other than the above can be utilized as a host provided that areplication origin for the recombinant DNA constructed herein and themutant AK gene make their function, the recombinant DNA can bereplicated, and the mutant AK activity can be enhanced. The mostpreferable host is AJ12036 strain (FERM P-7559) which is a wild typestrain of Corynebacterium glutamicum (Brevibacterium lactofermentum).

The transformant harboring the recombinant DNA containing the genecoding for aspartokinase in which synergistic feedback inhibition byL-lysine and L-threonine is substantially desensitized obtained by themethod described above is cultivated, and objective L-lysine is producedand accumulated in a culture liquid to collect it.

The medium to be used for L-lysine production is an ordinary mediumcontaining a carbon source, a nitrogen source, inorganic ions andoptionally other organic components.

As the carbon source, it is possible to use sugars such as glucose,lactose, galactose, fructose, and starch hydrolysate; alcohols such asglycerol and sorbitol; or organic acids such as fumaric acid, citricacid and succinic acid.

As the nitrogen source, it is possible to use inorganic ammonium saltssuch as ammonium sulfate, ammonium chloride and ammonium phosphate;organic nitrogen such as soybean hydrolysate; ammonia gas; and aqueousammonia.

It is desirable to allow required substances such as vitamin B₁ andL-homoserine or yeast extract to be contained in appropriate amounts asorganic trace nutrient sources. Other than the above, potassiumphosphate, magnesium sulfate, iron ion, manganese ion and so on areadded in small amounts, if necessary.

Cultivation is preferably carried out under an aerobic condition for16-72 hours. The cultivation temperature is controlled at 30° C. to 45°C., and pH is controlled at 5-7 during cultivation. Inorganic ororganic, acidic or alkaline substances as well as ammonia gas or thelike can be used for pH adjustment. Collection of L-lysine from acultivated liquor can be carried out by combining an ordinary ionexchange resin method, a precipitation method, and other known methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows steps of constructing p399AK9 and p399AKY from AK genefragments amplified from chromosome by PCR, and constructing p399AKYBand p399AK9B by introducing a DNA fragment (Coryne.-ori) having anability to allow plasmids to be autonomously proliferative in bacteriabelonging to the genus Corynebacterium. p399AK9B is constructed throughexactly the same steps as those for p399AKYB except that the former isdifferent from the latter in one base. Thus the both are shown togetherwith or without ( ).

FIG. 2 shows results of investigation on inhibition by lysine,threonine, and lysine+threonine of wild type and mutant (Thr) AK's. Theactivity obtained without addition of lysine and threonine was regardedas 100% to indicate the activity with addition thereof asactivity-holding ratios (desensitization degrees of inhibition).

FIG. 3 shows results of investigation on desensitization degrees ofinhibition by lysine for 8 kinds of AK's of the wild type and mutants.

FIG. 4 shows results of investigation on desensitization degrees ofinhibition by threonine for 8 kinds of AK's of the wild type andmutants.

FIG. 5 shows results of investigation on desensitization degrees ofconcerted inhibition by lysine+threonine for 8 kinds of AK's of the wildtype and mutants.

FIG. 6 shows results of investigation on thermal stability for 8 kindsof AK's of the wild type and mutants. The activity-holding ratio after atreatment at 55° C. for 90 minutes is indicated by %.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be concretely explained below with referenceto Examples.

Example 1 Preparation of Wild Type and Mutant AK Genes, and Preparationof Plasmids for Corynebacterium

Chromosomal DNA was prepared in accordance with an ordinary method fromCorynebacterium glutamicum (Brevibacterium lactofermentum) wild typestrain ATCC 13869, and L-lysine-producing mutant strain AJ3463 (FERMP-1987) obtained therefrom by a mutation treatment. AK genes wereamplified from the chromosomal DNA by PCR (polymerase chain reaction;see White, T. J. et al., Trends Genet., 5, 185 (1989)). For DNA primersused for the amplification, single strand DNA's of 23 mer and 21 merhaving sequences of 5'-TCGCGAAGTAGCACCTGTCACTT-3' (SEQ ID NO: 15) and5'-ACGGAATTCAATCTTACGGCC-3' (SEQ ID NO: 16) were synthesized to amplifya region of about 1,643 bp coding for the AK genes based on a sequenceknown for Corynebacterium glutamicum (see Molecular Microbiology (1991),5(5), 1197-1204; Mol. Gen. Genet. (1990), 224, 317-324). The DNA wassynthesized in accordance with an ordinary method using aphosphoramidite method (see Tetrahedron Letters (1981), 22, 1859) byusing a DNA synthesizer Model 380B produced by Applied Biosystems. Inthe PCR, the gene was amplified by using DNA Thermal Cycler Model PJ2000produced by Takara Shuzo Co., Ltd., using Taq DNA polymerase inaccordance with a method designated by the supplier. The amplified genefragment of 1,643 bp was confirmed by agarose gel electrophoresis.Subsequently, the fragment excised from the gel was purified inaccordance with an ordinary method, and digested with restrictionenzymes NruI (produced by Takara Shuzo) and EcoRI (produced by TakaraShuzo). pHSG399 (see Takeshita, S. et al., Gene (1987), 61, 63-74) wasused as a vector for cloning the gene fragment. pHSG399 was digestedwith restriction enzymes SmaI (produced by Takara Shuzo) and EcoRI, andligated with the amplified AK gene fragment. Ligation of DNA wasperformed by using DNA ligation kit (produced by Takara Shuzo) inaccordance with a designated method. Thus plasmids were prepared inwhich the AK gene fragments amplified from chromosome of Brevibacteriumwere ligated with pHSG399. A plasmid having the AK gene originating fromATCC 13869 as a wild type strain was designated as p399AKY, and aplasmid having the AK gene originating from AJ3463 as anL-lysine-producing bacterium was designated as p399AK9.

A DNA fragment having an ability to allow plasmids to be autonomouslyproliferative in bacteria belonging to the genus Corynebacterium(hereinafter referred to as "Coryne.-ori") was introduced into p399AKYand p399AK9 respectively to prepare plasmids carrying the AK genesautonomously replicable in bacteria belonging to the genusCorynebacterium. In order to obtain Coryne.-ori, a plasmid vector pHK4autonomously proliferative in bacterial cells of both Escherichia coliand bacteria belonging to the genus Corynebacterium was prepared. Someplasmid vectors autonomously proliferative in bacterial cells of bothEscherichia coli and bacteria belonging to the genus Corynebacterium hadbeen reported. Herein a novel shuttle vector pHK4 was constructed frompAJ1844 (see Japanese Patent Laid-open No. 58-216199) and pHSG298 (seeS. Takeshita et al., Gene, 61, 63-74 (1987)). pAJ1844 was partiallydigested with a restriction enzyme Sau3AI, and ligated with pHSG298having been completely digested with a restriction enzyme BamHI.Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ12036 (FERMP-7559) was transformed with DNA after the ligation. An electric pulsemethod (see Japanese Patent Laid-open No. 2-207791) was used fortransformation. Transformants were selected on M-CM2G plates containing25 μg/ml of kanamycin (containing glucose 5 g, polypeptone 10 g, yeastextract 10 g, NaCi 5 g, DL-methionine 0.2 g, and agar 15 g in 1 l ofpure water, pH 7.2). Plasmids were prepared from the transformants. Onehaving the smallest size was selected, and designated as pHK4. Thisplasmid is capable of autonomous proliferation in Escherichia coli andbacteria belonging to the genus Corynebacterium, and gives kanamycinresistance to the host.

pHK4 was digested with a restriction enzyme KpnI (produced by TakaraShuzo), and digested ends were blunt-ended. Blunt ends were formed byusing DNA Blunting kit (produced by Takara Shuzo) in accordance with adesignated method. After the blunt end formation, a phosphorylated BamHIlinker (produced by Takara Shuzo) was ligated to make modification sothat a DNA fragment of the Coryne.-ori portion could be excised frompHK4 by digestion only with BamHI. This plasmid was digested with BamHI.A generated Coryne.-ori DNA fragment was ligated with p399AKY andp399AK9 having been also digested with BamHI to prepare plasmids beingautonomously proliferative in bacteria belonging to the genusCorynebacterium and containing the AK genes. The plasmid containing thewild type AK gene originating from p399AKY was designated as p399AKYB,and the plasmid containing the mutant AK gene originating from p399AK9was designated as p399AK9B. Steps of constructing p399AK9B and p399AKYBare shown in FIG. 1. A strain AJ12691 obtained by introducing the mutantAK plasmid p399AK9B into AJ12086 strain (FERM P-7559) as a wild typestrain of Corynebacterium glutamicum (Brevibacterium divaricatum) hasbeen awarded a deposition number (FERM P-12918), and deposited inNational Institute of Bioscience and Human Technology of Agency ofIndustrial Science and Technology.

Example 2 Determination of Nucleotide Sequences of Wild Type AK andMutant AK Genes of Corynebacterium glutamicum

The plasmid p399AKY containing the wild type AK gene, and the plasmidp299AK9 containing the mutant AK gene were prepared to determinenucleotide sequences of the wild type and mutant AK genes. Nucleotidesequences were determined in accordance with a method of Sanger (F.Sanger et al., Proc. Natl. Acad. Sci., 74, 5463 (1977), etc.). Thenucleotide sequence of the wild type AK gene encoded by p399AKY is shownin SEQ ID NO: 1 in Sequence Listing. The nucleotide sequence of themutant AK gene encoded by p399AK9 is shown in SEQ ID NO: 2 in SequenceListing. The mutant AK gene has mutation of only one base such that1051th G is changed to A, as compared with the wild type AK. It is knownfor the AK gene that two subunits of α and β are encoded on an identicalDNA strand in an identical reading frame (see Kalinowski, J. et al.,Molecular Microbiology (1991) 5(5), 1197-1204). Judging from homology,it is also assumed for the genes of the present invention that twosubunits of α and β are encoded on an identical DNA strand in anidentical reading frame.

An amino acid sequence of the α-subunit of the wild type AK proteindeduced from the nucleotide sequence of DNA is shown in SEQ ID NO: 3 inSequence Listing. An amino acid sequence of the β-subunit is shown inSEQ ID NO: 5 in Sequence Listing. As those simultaneously showing theDNA sequence and the amino acid sequence, the α-subunit is shown in SEQID NO: 7 in Sequence Listing, and the β-subunit is shown in SEQ ID NO: 9in Sequence Listing. Nucleotide sequences corresponding to open readingframe portions of each of the α- and β-subunits are shown in SEQ ID NOS:11 and 13 in Sequence Listing, respectively.

Similarly, an amino acid sequence of the α-subunit of the mutant AKprotein deduced from the nucleotide sequence of DNA is shown in SEQ IDNO: 4 in Sequence Listing. An amino acid sequence of the β-subunit isshown in SEQ ID NO: 6 in Sequence Listing. As those simultaneouslyshowing the DNA sequence and the amino acid sequence, the α-subunit isshown in SEQ ID NO: 8 in Sequence Listing, and the β-subunit is shown inSEQ ID NO: 10 in Sequence Listing. Nucleotide sequences corresponding toopen reading frame portions of each of the α- and β-subunits are shownin SEQ ID NOS: 12 and 14 in Sequence Listing, respectively.

In each of the subunits, GTG is used as a start codon, and acorresponding amino acid is depicted as methionine. However, it isintended to represent methionine, valine, or formylmethionine. Themutation point of the mutant AK Gene implies that the mutant AK proteinundergoes amino acid substitution such that 279th alanine is changed tothreonine in the α-subunit, and 30th alanine is changed to threonine inthe β-subunit.

Example 3 Effect on L-Lysine Productivity by Introduction of Mutant AKand Wild Type AK Plasmids into Corynebacterium glutamicum Wild TypeStrain

Strains were prepared by introducing the wild type AK plasmid p399AKYBand the mutant AK plasmid p399AK9B respectively into AJ12036 strain(FERM P-7559) as a wild type strain of Corynebacterium glutamicum(Brevibacterium lactofermentum). The genes were introduced intoCorynebacterium by means of an electric pulse method. The aspartokinaseactivity was measured for Corynebacterium glutamicum (Brevibacteriumlactofermentum) AJ12036 strain as a host, AJ12690 strain harboring thewild type AK plasmid, and AJ12691 (FERM P12918) strain harboring themutant AK plasmid. The activity was measured in accordance with anordinary method (see Miyajima, R. et al., The Journal of Biochemistry(1968) 63(2), 139-148). As shown in Table 1, it was confirmed that theintroduction of the AK plasmid increased the specific activity of AKabout 10-15 times, and that the synergistic inhibition by L-lysine andL-threonine was desensitized only in the strain introduced with themutant AK plasmid. Table 1 shows specific activities of aspartokinasesof cell-disrupted solutions prepared from wild type AJ12036 strain ofCorynebacterium glutamicum (Brevibacterium lactofermentum), AJ12690strain additionally harboring the wild type AK plasmid, and AJ12691strain additionally harboring the mutant AK plasmid, and degrees ofsynergistic inhibition thereof by L-lysine and L-threonine. L-Lysine andL-threonine as inhibitors were added to give a final concentration of 1mM, respectively.

                  TABLE 1                                                         ______________________________________                                                  AK specific activity (mU/mg protein)                                Bacterial strain                                                                          No addition                                                                             +1 mM L-Lys, +1 mM L-Thr                                ______________________________________                                        AJ12036      19.0      2.6                                                    AJ12690     235.3      34.5                                                   AJ12691     210.5     145.3                                                   ______________________________________                                    

The lysine productivity was evaluated by cultivation for the wild typestrain AJ12036, the wild type AK plasmid-harboring strain AJ12690, andthe mutant AK plasmid-harboring strain AJ12691 (FERM P-12918). Theevaluation by cultivation was performed by inoculating the strains to aproduction medium (containing glucose 100 g, (NH₄)₂ SO₄ 55 g, KH₂ PO₄ 1g, MgSO₄.7H₂ O 1 g, soybean protein acid hydrolysate "Mamenou" (productof Ajinomoto Co., Ltd., trade name) 50 ml, FeSO₄.7H₂ O 10 mg, MnSO₄.4H₂O 10 mg, nicotinic acid amide 5 mg, and CaCO₃ 50 g in 1 l of pure water,pH 8.0), and cultivating them with reciprocatory shaking at 31.5° C. for72 hours. Amounts of produced lysine in culture liquids after thecultivation were as shown in Table 2. It is understood that the L-lysineproductivity is remarkably improved in the strain introduced with themutant AK plasmid. The plasmid-holding ratio upon completion of thecultivation was measured by using an index of chloramphenicol resistanceas a drug resistance marker of the plasmid. It was about 100%,exhibiting extremely high stability of the plasmid (Table 2). Table 2shows results of measurement of the L-lysine productivity byfermentation and the plasmid-holding ratio upon completion of thecultivation for wild type AJ12036 strain of Corynebacterium glutamicum(Brevibacterium lactofermentum), AJ12690 strain additionally harboringthe wild type AK plasmid, and AJ12691 strain additionally harboring themutant AK plasmid.

                  TABLE 2                                                         ______________________________________                                                    Amount of accumulated                                                                        Plasmid-holding                                    Bacterial strain                                                                          Lys (g/l)      ratio (%)                                          ______________________________________                                        AJ12036     0              --                                                 AJ12690     2              100                                                AJ12691     25              98                                                ______________________________________                                         --: no available data                                                    

Example 4 Analysis of Enzymes of Wild Type AK and Mutant AK ofCorynebacterium glutamicum

For measurement and evaluation of the enzyme activity of AK, anAK-completely deficient strain of Escherichia coli Gif106M1 was used asa host (Boy, E. and Patte, J. C., J. Bacteriol. 112, 84-92 (1972),Theze, J. et al., J. Bacteriol. 117, 133-143 (1974)), otherwise AK of ahost and AK from a plasmid might exist in a mixed manner, resulting inimpossibility to perform correct measurement, because no AK-deficientstrain was present in bacteria belonging to the genus Corynebacterium.It is known that most of genes of bacteria belonging to the genusCorynebacterium are expressed in Escherichia coli. In addition, the AKgene was ligated at a position downstream from lac promoter on pHSG399.Thus it was postulated that the gene could be expressed in Escherichiacoli.

At first, Gif106M1 was transformed with p399AKY and p399AK9 prepared inExample 1 to confirm growth complementation in a minimum medium M9 shownbelow. Thus it was confirmed that AK from the bacteria belonging to thegenus Corynebacterium was expressed and operated in cells of Escherichiacoli.

Minimum medium M9

A: 20×M9

Na₂ HPO₄.12H₂ O 303 (g/L)

KH₂ PO₄ 60

NaCl 10

NH₄ Cl 20

B: 1M MgSO₄

C: 50% Glucose

D: 1 g/L Thiamine

A, B, C, and D were sterilized separately, and mixed in a ratio ofA:B:C:D=5:0.1:1:0.1:95.

Subsequently, cell-free extracts were prepared from bacterial cells, andthe enzyme activity of AK was measured.

Upon measurement of the enzyme activity of AK, various concentrations oflysine and threonine were added to enzyme reaction solutions toinvestigate the degree of inhibition (FIG. 2). As a result, it was foundthat the mutant AK exhibited little improvement in inhibition by lysinealone as compared with the wild type, however, inhibition by threoninewas desensitized by 100% even with activation a little, and that thedesensitization of inhibition by threonine resulted in mitigation ofconcerted inhibition by lysine+threonine. (Ki value: 0.4 mM→5.0 mM).

Example 5 Preparation of Inhibition-Desensitized Type AK Gene ofCorynebacterium glutamicum

It was revealed from Example 4 that the mutant AK was insufficient indesensitization of inhibition by lysine alone. Thus it was intended toimprove this property by introducing mutation.

Site-directed mutagenesis was used as a method for preparinginhibition-desensitized type AK genes. It was intended to substitute themutation point specified in Example 2 (²⁷⁹ Ala→Thr) with other aminoacid residues. The site-directed mutagenesis method for causingobjective mutation at an objective site includes, for example, a methodusing PCR (Higuchi, R., 61, in PCR Technology (Erlich, H. A. Eds.,Stockton press (1989)), and a method using phage (Kramer, W. and Frits,H. J., Meth. in Enzymol., 154 350 (1987); Kunkel, T. A. et al., Meth. inEnzymol., 154 367 (1987)).

Types of amino acid residues to be introduced by mutation were asfollows. Twenty kinds of amino acids were classified according toseveral properties such as polarity and molecular structure, and 8 kindsof representatives (Arg, Asp, Cys, Phe, Pro, Ser, Tyr, Val) wereselected. Amino acid mutation and nucleotide substitution at themutation point of each of them are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                                 Name of plasmid                                                               (name after                                          Name of  Mutation point and                                                                            introduction of                                      mutation amino acid change                                                                             Coryne.-ori)                                         ______________________________________                                        Wild type                p399AKY (P399AKYB)                                   Thr      .sup.279 Ala GCT → Thr A*CT                                                            p399AK9 (p399AK9B)                                   Arg      .sup.279 Ala GCT → Arg C*G*T                                                           p399AKAR (p399AKARB)                                 Asp      .sup.279 Ala GCT → Asp GA*T                                                            p399AKAD                                             Cys      .sup.279 Ala GCT → Cys T*G*T                                                           p399AKAC (p399AKACB)                                 Phe      .sup.279 Ala GCT → Phe T*T*T                                                           p399AKAF (p399AKAFB)                                 Pro      .sup.279 Ala GCT → Pro C*CT                                                            p399AKAP                                             (p399AKAPB)                                                                   Ser      .sup.279 Ala GCT → Ser T*CT                                                            p399AKAS                                             (p399AKASB)                                                                   Tyr      .sup.279 Ala GCT → Tyr T*A*T                                                           p399AKAY (p399AKAYB)                                 Val      .sup.279 Ala GCT → Val GT*T                                                            p399AKAV                                             (p399AKAVB)                                                                   ______________________________________                                    

A method for introducing mutation was as follows. Eight species ofsynthetic DNA of 23 mer were designed, in which the codon of the 279thAla residue for introducing mutation was substituted with the codons ofthe objective amino acid residues. Synthetic DNA for introducing Arg was5'-GCCAGGCGAG CGT GCCAAGGTTT-3' (SEQ ID NO: 17), synthetic DNA forintroducing Asp was 5'-GCCAGGCGAG GAT GCCAAGGTTT-3' (SEQ ID NO: 18),synthetic DNA for introducing Cys was 5'-GCCAGGCGAG TGT GCCAAGGTTT-3'(SEQ ID NO: 19), synthetic DNA for introducing Phe was 5'-GCCAGGCGAG TTTGCCAAGGTTT-3' (SEQ ID NO: 20), synthetic DNA for introducing Pro was5'-GCCAGGCGAG CCT GCCAAGGTTT-3' (SEQ ID NO: 21), synthetic DNA forintroducing Ser was 5'-GCCAGGCGAG TCT GCCAAGGTTT-3' (SEQ ID NO: 22),synthetic DNA for introducing Tyr was 5'-GCCAGGCGAG TAT GCCAAGGTTT-3'(SEQ ID NO: 23), and synthetic DNA for introducing Val was 5'-GCCAGGCGAGGTT GCCAAGGTTT-3' (SEQ ID NO: 24). Sixteen species of 23 mer singlestrand DNA's were synthesized together with complementary sequencesthereof. For example, when the Arg residue was introduced, the singlestrand DNA having the sequence of 5'-GCCAGGCGAG CGT GCCAAGGTTT-3'(SEQ IDNO:17), the single strand DNA complementary thereto, the single strandDNA having the sequence of SEQ ID NO: 15, and the single strand DNAhaving the sequence of SEQ ID NO: 16 were used as primers to conduct thePCR method by using pB99AKY as a template. In order to excludeintroduction of non-specific mutation, about 280 base pairs containingthe mutation point were excised from prepared DNA by using restrictionenzymes (NaeI-AvaII) to make substitution with a corresponding portionof p399AKY. The nucleotide sequence was confirmed for the substitutedregion.

Example 6 Analysis of 8 Kinds of Enzymes of Mutant AK Genes

Gif106M1 was transformed with 8 kinds of the plasmids containing each ofthe mutant AK genes obtained in Example 5 in accordance with a methodsimilar to that in Example 4. Cell-free extracts were prepared, andenzymes were analyzed. The degree of desensitization of inhibition, andthe specific activity upon addition of lysine 5 mM, threonine 5 mM, orlysine 5 mM+threonine 5 mM are shown in Table 4. The degree ofdesensitization of inhibition upon addition of each concentration oflysine and/or threonine is graphically shown in FIGS. 3, 4 and 5.

                  TABLE 4                                                         ______________________________________                                                Specific                                                                      activity                                                                      (mU/mg                                                                              5 mM Lys   5 mM Thr 5 mM Lys                                            protein)                                                                            (%)        (%)      + Thr (%)                                   ______________________________________                                        Wild type 15.3    42.3        62.3   9.2                                      Thr       12.9    47.0       103.5  50.4                                      Pro        2.8    76.9       126.9  103.8                                     Cys       15.4    56.3       108.1  17.0                                      Ser        8.2    52.6       131.6  18.4                                      Val       21.8    51.1        98.3  52.3                                      Arg        7.6    40.6       107.2  47.8                                      Tyr       14.4    14.4       103.6  19.4                                      Phe       18.7    12.1       103.0  18.2                                      Asp        1.5    --         --     --                                        ______________________________________                                    

AK was inactivated in the case of change to acidic amino acid such asAsp. However, the inhibition by threonine was desensitized in the caseof change to any other amino acid. The mutation could be generallyclassified into 4 groups for other properties. Mutation similar to themutant (Thr) in Example 2 includes the Val residue-introduced mutantstrain, and the Arg residue-introduced mutant strain. As for the Cysresidue-introduced mutant strain and the Ser residue-introduced mutantstrain, the inhibition by lysine alone was equivalent to that in thewild type, however, the inhibition was consequently enhanced in the caseof the concerted inhibition. The concerted inhibition has been enhancedprobably because the behavior to threonine has a characteristic propertyto give a crest-shaped graph such that activation occurs at lowconcentrations but inhibition occurs at high concentrations. Theinhibition by lysine alone Was enhanced in the Phe residue-introducedmutant strain and the Tyr residue-introduced mutant strain concerningaromatic amino acids, as compared with the wild type. The Proresidue-introduced mutant strain had a low specific activity (about 1/5of that of the wild type) probably because Pro might greatly affect thestereochemical structure. However, the inhibition by lysine alone wasmitigated, and the degree of activation by threonine became large (notless than 120%). Thus the concerted inhibition was also desensitized.

Thermal stability was investigated as an index of stability of theenzyme structure constructed by the introduction of mutation. Thetreatment condition was set at 55° C. for 1.5 hour in which the activityof the wild type AK was about 80%. The Cys residue-introduced mutantstrain, the Thr residue-introduced mutant strain, the Pheresidue-introduced mutant strain, the Tyr residue-introduced mutantstrain, and the Val residue-introduced mutant strain had higherstability than that of the wild type, among which the Valresidue-introduced mutant strain had the highest stability (FIG. 6).

Example 7 Effect on L-Lysine Productivity by Introduction of PlasmidsContaining 8 Kinds of Mutant AK Genes and Wild Type AK Gene intoCorynebacterium glutamicum Wild type strain

Strains were prepared by introducing 8 kinds of the plasmids shown inTable 3 into AJ12036 strain (FERM P-7559) as a wild type strain ofCorynebacterium glutamicum (Brevibacterium lactofermentum) in the samemanner as Example 3. The AK activity was measured for each of thestrains. As shown in Table 5, the AK specific activities of the strainsintroduced with each of the plasmids were increased about 20-80 times ascompared with the AK specific activity of the host. The desensitizationdegree of inhibition by lysine and/or threonine was similar to those inExample 6. The Pro residue-introduced mutant AK had the highest degreeof desensitization of inhibition, in which the degree of desensitizationof inhibition was greater than that of the Thr reside-introduced mutantAK, with respect to any of the inhibition by lysine alone, theinhibition by threonine alone, and the concerted inhibition by the both.

                  TABLE 5                                                         ______________________________________                                                Specific                                                                      activity                                                                      (mU/mg                                                                              5 mM Lys   5 mM Thr 2 mM Lys                                            protein)                                                                            (%)        (%)      + Thr (%)                                   ______________________________________                                        AJ12036    5.6    52.0        82.0   7.0                                      Wild type 316.4   52.7        86.8   6.2                                      Thr       374.4   58.7       109.1  78.3                                      Arg       197.4   41.4       106.8  58.6                                      Cys       267.0   66.5       135.7  60.6                                      Phe       447.7   14.6       105.0  32.4                                      Pro       125.0   77.5       123.2  85.2                                      Ser       406.8   55.0       114.4  37.0                                      Tyr       425.6   16.1       104.8  32.2                                      Val       448.9   60.5       103.5  75.5                                      ______________________________________                                    

The lysine productivity was further evaluated by cultivation for the 9kinds of these strains by using a method similar to that in Example 3.The amount of produced lysine in each cultivation is as shown in Table6. It is understood that the L-lysine productivity is remarkablyimproved by introducing the mutant AK plasmids. Especially, highaccumulation of about 25 g/l was exhibited in the mutant strains otherthan the Cys residue-introduced mutant strain and the Setresidue-introduced mutant strain. The plasmid-holding ratio uponcompletion of the cultivation was approximately 100%, exhibiting highstability of the plasmids.

                  TABLE 6                                                         ______________________________________                                                 Lys--HCl (g/l)                                                                         Plasmid holding ratio (%)                                   ______________________________________                                        Wild type   0.00      100                                                     Thr        24.25      100                                                     Arg        24.56      100                                                     Cys        13.41      100                                                     Phe        25.14      100                                                     Pro        25.11      100                                                     Ser         5.72      100                                                     Tyr        25.12      100                                                     Val        25.02      100                                                     ______________________________________                                    

Industrial Applicability

In the AK gene of Brevibacterium lactofermentum, Ala located at theposition 279 in accordance with the amino acid numbers in SEQ ID NO: 3or at the position 30 in accordance with the amino acid numbers in SEQID NO:5 was changed to amino acids other than acidic amino acids. ThusAK was obtained in which inhibition by threonine was completelydesensitized, and consequently concerted inhibition by lysine+threoninewas desensitized. Especially, AK was obtained, in which inhibition bylysine alone was partially desensitized, by changing the amino acidresidue at the aforementioned position to Pro. AK having improvedthermal stability was obtained by changing the aforementioned site toVal, Tyr, or Phe. The productivity of L-lysine could be remarkablyincreased by increasing the activity of such mutant AK in cells ofcoryneform bacteria.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 24                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1643 nucleotides                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: ATCC 13869                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TCGCGAAGTAGCACCTGTCACTTTTGTCTCAAATATTAAATCGAATATCAATATACGGTC60                TGTTTATTGGAACGCATCCCAGTGGCTGAGACGCATCCGCTAAAGCCCCAGGAACCCTGT120               GCAGAAAGAAAACACTCCTCTGGCTAGGTAGACACAGTTTATAAAGGTAGAGTTGAGCGG180               GTAACTGTCAGCACGTAGATCGAAAGGTGCACAAAGGTGGCCCTGGTCGTACAGAAATAT240               GGCGGTTCCTCGCTTGAGAGTGCGGAACGCATTAGAAACGTCGCTGAACGGATCGTTGCC300               ACCAAGAAGGCTGGAAATGATGTCGTGGTTGTCTGCTCCGCAATGGGAGACACCACGGAT360               GAACTTCTAGAACTTGCAGCGGCAGTGAATCCCGTTCCGCCAGCTCGTGAAATGGATATG420               CTCCTGACTGCTGGTGAGCGTATTTCTAACGCTCTCGTCGCCATGGCTATTGAGTCCCTT480               GGCGCAGAAGCTCAATCTTTCACTGGCTCTCAGGCTGGTGTGCTCACCACCGAGCGCCAC540               GGAAACGCACGCATTGTTGACGTCACACCGGGTCGTGTGCGTGAAGCACTCGATGAGGGC600               AAGATCTGCATTGTTGCTGGTTTTCAGGGTGTTAATAAAGAAACCCGCGATGTCACCACG660               TTGGGTCGTGGTGGTTCTGACACCACTGCAGTTGCGTTGGCAGCTGCTTTGAACGCTGAT720               GTGTGTGAGATTTACTCGGACGTTGACGGTGTGTATACCGCTGACCCGCGCATCGTTCCT780               AATGCACAGAAGCTGGAAAAGCTCAGCTTCGAAGAAATGCTGGAACTTGCTGCTGTTGGC840               TCCAAGATTTTGGTGCTGCGCAGTGTTGAATACGCTCGTGCATTCAATGTGCCACTTCGC900               GTACGCTCGTCTTATAGTAATGATCCCGGCACTTTGATTGCCGGCTCTATGGAGGATATT960               CCTGTGGAAGAAGCAGTCCTTACCGGTGTCGCAACCGACAAGTCCGAAGCCAAAGTAACC1020              GTTCTGGGTATTTCCGATAAGCCAGGCGAGGCTGCCAAGGTTTTCCGTGCGTTGGCTGAT1080              GCAGAAATCAACATTGACATGGTTCTGCAGAACGTCTCCTCTGTGGAAGACGGCACCACC1140              GACATCACGTTCACCTGCCCTCGCGCTGACGGACGCCGTGCGATGGAGATCTTGAAGAAG1200              CTTCAGGTTCAGGGCAACTGGACCAATGTGCTTTACGACGACCAGGTCGGCAAAGTCTCC1260              CTCGTGGGTGCTGGCATGAAGTCTCACCCAGGTGTTACCGCAGAGTTCATGGAAGCTCTG1320              CGCGATGTCAACGTGAACATCGAATTGATTTCCACCTCTGAGATCCGCATTTCCGTGCTG1380              ATCCGTGAAGATGATCTGGATGCTGCTGCACGTGCATTGCATGAGCAGTTCCAGCTGGGC1440              GGCGAAGACGAAGCCGTCGTTTATGCAGGCACCGGACGCTAAAGTTTTAAAGGAGTAGTT1500              TTACAATGACCACCATCGCAGTTGTTGGTGCAACCGGCCAGGTCGGCCAGGTTATGCGCA1560              CCCTTTTGGAAGAGCGCAATTTCCCAGCTGACACTGTTCGTTTCTTTGCTTCCCCGCGTT1620              CCGCAGGCCGTAAGATTGAATTC1643                                                   (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1643 nucleotides                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: AJ3463                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TCGCGAAGTAGCACCTGTCACTTTTGTCTCAAATATTAAATCGAATATCAATATACGGTC60                TGTTTATTGGAACGCATCCCAGTGGCTGAGACGCATCCGCTAAAGCCCCAGGAACCCTGT120               GCAGAAAGAAAACACTCCTCTGGCTAGGTAGACACAGTTTATAAAGGTAGAGTTGAGCGG180               GTAACTGTCAGCACGTAGATCGAAAGGTGCACAAAGGTGGCCCTGGTCGTACAGAAATAT240               GGCGGTTCCTCGCTTGAGAGTGCGGAACGCATTAGAAACGTCGCTGAACGGATCGTTGCC300               ACCAAGAAGGCTGGAAATGATGTCGTGGTTGTCTGCTCCGCAATGGGAGACACCACGGAT360               GAACTTCTAGAACTTGCAGCGGCAGTGAATCCCGTTCCGCCAGCTCGTGAAATGGATATG420               CTCCTGACTGCTGGTGAGCGTATTTCTAACGCTCTCGTCGCCATGGCTATTGAGTCCCTT480               GGCGCAGAAGCTCAATCTTTCACTGGCTCTCAGGCTGGTGTGCTCACCACCGAGCGCCAC540               GGAAACGCACGCATTGTTGACGTCACACCGGGTCGTGTGCGTGAAGCACTCGATGAGGGC600               AAGATCTGCATTGTTGCTGGTTTTCAGGGTGTTAATAAAGAAACCCGCGATGTCACCACG660               TTGGGTCGTGGTGGTTCTGACACCACTGCAGTTGCGTTGGCAGCTGCTTTGAACGCTGAT720               GTGTGTGAGATTTACTCGGACGTTGACGGTGTGTATACCGCTGACCCGCGCATCGTTCCT780               AATGCACAGAAGCTGGAAAAGCTCAGCTTCGAAGAAATGCTGGAACTTGCTGCTGTTGGC840               TCCAAGATTTTGGTGCTGCGCAGTGTTGAATACGCTCGTGCATTCAATGTGCCACTTCGC900               GTACGCTCGTCTTATAGTAATGATCCCGGCACTTTGATTGCCGGCTCTATGGAGGATATT960               CCTGTGGAAGAAGCAGTCCTTACCGGTGTCGCAACCGACAAGTCCGAAGCCAAAGTAACC1020              GTTCTGGGTATTTCCGATAAGCCAGGCGAGACTGCCAAGGTTTTCCGTGCGTTGGCTGAT1080              GCAGAAATCAACATTGACATGGTTCTGCAGAACGTCTCCTCTGTGGAAGACGGCACCACC1140              GACATCACGTTCACCTGCCCTCGCGCTGACGGACGCCGTGCGATGGAGATCTTGAAGAAG1200              CTTCAGGTTCAGGGCAACTGGACCAATGTGCTTTACGACGACCAGGTCGGCAAAGTCTCC1260              CTCGTGGGTGCTGGCATGAAGTCTCACCCAGGTGTTACCGCAGAGTTCATGGAAGCTCTG1320              CGCGATGTCAACGTGAACATCGAATTGATTTCCACCTCTGAGATCCGCATTTCCGTGCTG1380              ATCCGTGAAGATGATCTGGATGCTGCTGCACGTGCATTGCATGAGCAGTTCCAGCTGGGC1440              GGCGAAGACGAAGCCGTCGTTTATGCAGGCACCGGACGCTAAAGTTTTAAAGGAGTAGTT1500              TTACAATGACCACCATCGCAGTTGTTGGTGCAACCGGCCAGGTCGGCCAGGTTATGCGCA1560              CCCTTTTGGAAGAGCGCAATTTCCCAGCTGACACTGTTCGTTTCTTTGCTTCCCCGCGTT1620              CCGCAGGCCGTAAGATTGAATTC1643                                                   (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 421 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: ATCC13869                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetAlaLeuValValGlnLysTyrGlyGlySerSerLeuGluSerAla                              151015                                                                        GluArgIleArgAsnValAlaGluArgIleValAlaThrLysLysAla                              202530                                                                        GlyAsnAspValValValValCysSerAlaMetGlyAspThrThrAsp                              354045                                                                        GluLeuLeuGluLeuAlaAlaAlaValAsnProValProProAlaArg                              505560                                                                        GluMetAspMetLeuLeuThrAlaGlyGluArgIleSerAsnAlaLeu                              65707580                                                                      ValAlaMetAlaIleGluSerLeuGlyAlaGluAlaGlnSerPheThr                              859095                                                                        GlySerGlnAlaGlyValLeuThrThrGluArgHisGlyAsnAlaArg                              100105110                                                                     IleValAspValThrProGlyArgValArgGluAlaLeuAspGluGly                              115120125                                                                     LysIleCysIleValAlaGlyPheGlnGlyValAsnLysGluThrArg                              130135140                                                                     AspValThrThrLeuGlyArgGlyGlySerAspThrThrAlaValAla                              145150155160                                                                  LeuAlaAlaAlaLeuAsnAlaAspValCysGluIleTyrSerAspVal                              165170175                                                                     AspGlyValTyrThrAlaAspProArgIleValProAsnAlaGlnLys                              180185190                                                                     LeuGluLysLeuSerPheGluGluMetLeuGluLeuAlaAlaValGly                              195200205                                                                     SerLysIleLeuValLeuArgSerValGluTyrAlaArgAlaPheAsn                              210215220                                                                     ValProLeuArgValArgSerSerTyrSerAsnAspProGlyThrLeu                              225230235240                                                                  IleAlaGlySerMetGluAspIleProValGluGluAlaValLeuThr                              245250255                                                                     GlyValAlaThrAspLysSerGluAlaLysValThrValLeuGlyIle                              260265270                                                                     SerAspLysProGlyGluAlaAlaLysValPheArgAlaLeuAlaAsp                              275280285                                                                     AlaGluIleAsnIleAspMetValLeuGlnAsnValSerSerValGlu                              290295300                                                                     AspGlyThrThrAspIleThrPheThrCysProArgAlaAspGlyArg                              305310315320                                                                  ArgAlaMetGluIleLeuLysLysLeuGlnValGlnGlyAsnTrpThr                              325330335                                                                     AsnValLeuTyrAspAspGlnValGlyLysValSerLeuValGlyAla                              340345350                                                                     GlyMetLysSerHisProGlyValThrAlaGluPheMetGluAlaLeu                              355360365                                                                     ArgAspValAsnValAsnIleGluLeuIleSerThrSerGluIleArg                              370375380                                                                     IleSerValLeuIleArgGluAspAspLeuAspAlaAlaAlaArgAla                              385390395400                                                                  LeuHisGluGlnPheGlnLeuGlyGlyGluAspGluAlaValValTyr                              405410415                                                                     AlaGlyThrGlyArg                                                               420                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 421 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: AJ3463                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAlaLeuValValGlnLysTyrGlyGlySerSerLeuGluSerAla                              151015                                                                        GluArgIleArgAsnValAlaGluArgIleValAlaThrLysLysAla                              202530                                                                        GlyAsnAspValValValValCysSerAlaMetGlyAspThrThrAsp                              354045                                                                        GluLeuLeuGluLeuAlaAlaAlaValAsnProValProProAlaArg                              505560                                                                        GluMetAspMetLeuLeuThrAlaGlyGluArgIleSerAsnAlaLeu                              65707580                                                                      ValAlaMetAlaIleGluSerLeuGlyAlaGluAlaGlnSerPheThr                              859095                                                                        GlySerGlnAlaGlyValLeuThrThrGluArgHisGlyAsnAlaArg                              100105110                                                                     IleValAspValThrProGlyArgValArgGluAlaLeuAspGluGly                              115120125                                                                     LysIleCysIleValAlaGlyPheGlnGlyValAsnLysGluThrArg                              130135140                                                                     AspValThrThrLeuGlyArgGlyGlySerAspThrThrAlaValAla                              145150155160                                                                  LeuAlaAlaAlaLeuAsnAlaAspValCysGluIleTyrSerAspVal                              165170175                                                                     AspGlyValTyrThrAlaAspProArgIleValProAsnAlaGlnLys                              180185190                                                                     LeuGluLysLeuSerPheGluGluMetLeuGluLeuAlaAlaValGly                              195200205                                                                     SerLysIleLeuValLeuArgSerValGluTyrAlaArgAlaPheAsn                              210215220                                                                     ValProLeuArgValArgSerSerTyrSerAsnAspProGlyThrLeu                              225230235240                                                                  IleAlaGlySerMetGluAspIleProValGluGluAlaValLeuThr                              245250255                                                                     GlyValAlaThrAspLysSerGluAlaLysValThrValLeuGlyIle                              260265270                                                                     SerAspLysProGlyGluThrAlaLysValPheArgAlaLeuAlaAsp                              275280285                                                                     AlaGluIleAsnIleAspMetValLeuGlnAsnValSerSerValGlu                              290295300                                                                     AspGlyThrThrAspIleThrPheThrCysProArgAlaAspGlyArg                              305310315320                                                                  ArgAlaMetGluIleLeuLysLysLeuGlnValGlnGlyAsnTrpThr                              325330335                                                                     AsnValLeuTyrAspAspGlnValGlyLysValSerLeuValGlyAla                              340345350                                                                     GlyMetLysSerHisProGlyValThrAlaGluPheMetGluAlaLeu                              355360365                                                                     ArgAspValAsnValAsnIleGluLeuIleSerThrSerGluIleArg                              370375380                                                                     IleSerValLeuIleArgGluAspAspLeuAspAlaAlaAlaArgAla                              385390395400                                                                  LeuHisGluGlnPheGlnLeuGlyGlyGluAspGluAlaValValTyr                              405410415                                                                     AlaGlyThrGlyArg                                                               420                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 172 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: ATCC13869                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetGluGluAlaValLeuThrGlyValAlaThrAspLysSerGluAla                              151015                                                                        LysValThrValLeuGlyIleSerAspLysProGlyGluAlaAlaLys                              202530                                                                        ValPheArgAlaLeuAlaAspAlaGluIleAsnIleAspMetValLeu                              354045                                                                        GlnAsnValSerSerValGluAspGlyThrThrAspIleThrPheThr                              505560                                                                        CysProArgAlaAspGlyArgArgAlaMetGluIleLeuLysLysLeu                              65707580                                                                      GlnValGlnGlyAsnTrpThrAsnValLeuTyrAspAspGlnValGly                              859095                                                                        LysValSerLeuValGlyAlaGlyMetLysSerHisProGlyValThr                              100105110                                                                     AlaGluPheMetGluAlaLeuArgAspValAsnValAsnIleGluLeu                              115120125                                                                     IleSerThrSerGluIleArgIleSerValLeuIleArgGluAspAsp                              130135140                                                                     LeuAspAlaAlaAlaArgAlaLeuHisGluGlnPheGlnLeuGlyGly                              145150155160                                                                  GluAspGluAlaValValTyrAlaGlyThrGlyArg                                          165170                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 172 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: AJ3463                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetGluGluAlaValLeuThrGlyValAlaThrAspLysSerGluAla                              151015                                                                        LysValThrValLeuGlyIleSerAspLysProGlyGluThrAlaLys                              202530                                                                        ValPheArgAlaLeuAlaAspAlaGluIleAsnIleAspMetValLeu                              354045                                                                        GlnAsnValSerSerValGluAspGlyThrThrAspIleThrPheThr                              505560                                                                        CysProArgAlaAspGlyArgArgAlaMetGluIleLeuLysLysLeu                              65707580                                                                      GlnValGlnGlyAsnTrpThrAsnValLeuTyrAspAspGlnValGly                              859095                                                                        LysValSerLeuValGlyAlaGlyMetLysSerHisProGlyValThr                              100105110                                                                     AlaGluPheMetGluAlaLeuArgAspValAsnValAsnIleGluLeu                              115120125                                                                     IleSerThrSerGluIleArgIleSerValLeuIleArgGluAspAsp                              130135140                                                                     LeuAspAlaAlaAlaArgAlaLeuHisGluGlnPheGlnLeuGlyGly                              145150155160                                                                  GluAspGluAlaValValTyrAlaGlyThrGlyArg                                          165170                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1643 nucleotides                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: ATCC13869                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat peptide                                                     (B) LOCATION: 217..1482                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TCGCGAAGTAGCACCTGTCACTTTTGTCTCAAATATTAAATCGAATATCAATATACGGTC60                TGTTTATTGGAACGCATCCCAGTGGCTGAGACGCATCCGCTAAAGCCCCAGGAACCCTGT120               GCAGAAAGAAAACACTCCTCTGGCTAGGTAGACACAGTTTATAAAGGTAGAGTTGAGCGG180               GTAACTGTCAGCACGTAGATCGAAAGGTGCACAAAGGTGGCCCTGGTCGTACAG234                     MetAlaLeuValValGln                                                            15                                                                            AAATATGGCGGTTCCTCGCTTGAGAGTGCGGAACGCATTAGAAACGTC282                           LysTyrGlyGlySerSerLeuGluSerAlaGluArgIleArgAsnVal                              101520                                                                        GCTGAACGGATCGTTGCCACCAAGAAGGCTGGAAATGATGTCGTGGTT330                           AlaGluArgIleValAlaThrLysLysAlaGlyAsnAspValValVal                              253035                                                                        GTCTGCTCCGCAATGGGAGACACCACGGATGAACTTCTAGAACTTGCA378                           ValCysSerAlaMetGlyAspThrThrAspGluLeuLeuGluLeuAla                              404550                                                                        GCGGCAGTGAATCCCGTTCCGCCAGCTCGTGAAATGGATATGCTCCTG426                           AlaAlaValAsnProValProProAlaArgGluMetAspMetLeuLeu                              55606570                                                                      ACTGCTGGTGAGCGTATTTCTAACGCTCTCGTCGCCATGGCTATTGAG474                           ThrAlaGlyGluArgIleSerAsnAlaLeuValAlaMetAlaIleGlu                              758085                                                                        TCCCTTGGCGCAGAAGCTCAATCTTTCACTGGCTCTCAGGCTGGTGTG522                           SerLeuGlyAlaGluAlaGlnSerPheThrGlySerGlnAlaGlyVal                              9095100                                                                       CTCACCACCGAGCGCCACGGAAACGCACGCATTGTTGACGTCACACCG570                           LeuThrThrGluArgHisGlyAsnAlaArgIleValAspValThrPro                              105110115                                                                     GGTCGTGTGCGTGAAGCACTCGATGAGGGCAAGATCTGCATTGTTGCT618                           GlyArgValArgGluAlaLeuAspGluGlyLysIleCysIleValAla                              120125130                                                                     GGTTTTCAGGGTGTTAATAAAGAAACCCGCGATGTCACCACGTTGGGT666                           GlyPheGlnGlyValAsnLysGluThrArgAspValThrThrLeuGly                              135140145150                                                                  CGTGGTGGTTCTGACACCACTGCAGTTGCGTTGGCAGCTGCTTTGAAC714                           ArgGlyGlySerAspThrThrAlaValAlaLeuAlaAlaAlaLeuAsn                              155160165                                                                     GCTGATGTGTGTGAGATTTACTCGGACGTTGACGGTGTGTATACCGCT762                           AlaAspValCysGluIleTyrSerAspValAspGlyValTyrThrAla                              170175180                                                                     GACCCGCGCATCGTTCCTAATGCACAGAAGCTGGAAAAGCTCAGCTTC810                           AspProArgIleValProAsnAlaGlnLysLeuGluLysLeuSerPhe                              185190195                                                                     GAAGAAATGCTGGAACTTGCTGCTGTTGGCTCCAAGATTTTGGTGCTG858                           GluGluMetLeuGluLeuAlaAlaValGlySerLysIleLeuValLeu                              200205210                                                                     CGCAGTGTTGAATACGCTCGTGCATTCAATGTGCCACTTCGCGTACGC906                           ArgSerValGluTyrAlaArgAlaPheAsnValProLeuArgValArg                              215220225230                                                                  TCGTCTTATAGTAATGATCCCGGCACTTTGATTGCCGGCTCTATGGAG954                           SerSerTyrSerAsnAspProGlyThrLeuIleAlaGlySerMetGlu                              235240245                                                                     GATATTCCTGTGGAAGAAGCAGTCCTTACCGGTGTCGCAACCGACAAG1002                          AspIleProValGluGluAlaValLeuThrGlyValAlaThrAspLys                              250255260                                                                     TCCGAAGCCAAAGTAACCGTTCTGGGTATTTCCGATAAGCCAGGCGAG1050                          SerGluAlaLysValThrValLeuGlyIleSerAspLysProGlyGlu                              265270275                                                                     GCTGCCAAGGTTTTCCGTGCGTTGGCTGATGCAGAAATCAACATTGAC1098                          AlaAlaLysValPheArgAlaLeuAlaAspAlaGluIleAsnIleAsp                              280285290                                                                     ATGGTTCTGCAGAACGTCTCCTCTGTGGAAGACGGCACCACCGACATC1146                          MetValLeuGlnAsnValSerSerValGluAspGlyThrThrAspIle                              295300305310                                                                  ACGTTCACCTGCCCTCGCGCTGACGGACGCCGTGCGATGGAGATCTTG1194                          ThrPheThrCysProArgAlaAspGlyArgArgAlaMetGluIleLeu                              315320325                                                                     AAGAAGCTTCAGGTTCAGGGCAACTGGACCAATGTGCTTTACGACGAC1242                          LysLysLeuGlnValGlnGlyAsnTrpThrAsnValLeuTyrAspAsp                              330335340                                                                     CAGGTCGGCAAAGTCTCCCTCGTGGGTGCTGGCATGAAGTCTCACCCA1290                          GlnValGlyLysValSerLeuValGlyAlaGlyMetLysSerHisPro                              345350355                                                                     GGTGTTACCGCAGAGTTCATGGAAGCTCTGCGCGATGTCAACGTGAAC1338                          GlyValThrAlaGluPheMetGluAlaLeuArgAspValAsnValAsn                              360365370                                                                     ATCGAATTGATTTCCACCTCTGAGATCCGCATTTCCGTGCTGATCCGT1386                          IleGluLeuIleSerThrSerGluIleArgIleSerValLeuIleArg                              375380385390                                                                  GAAGATGATCTGGATGCTGCTGCACGTGCATTGCATGAGCAGTTCCAG1434                          GluAspAspLeuAspAlaAlaAlaArgAlaLeuHisGluGlnPheGln                              395400405                                                                     CTGGGCGGCGAAGACGAAGCCGTCGTTTATGCAGGCACCGGACGCTAA1482                          LeuGlyGlyGluAspGluAlaValValTyrAlaGlyThrGlyArg                                 410415420421                                                                  AGTTTTAAAGGAGTAGTTTTACAATGACCACCATCGCAGTTGTTGGTGCAACCGGCCAGG1542              TCGGCCAGGTTATGCGCACCCTTTTGGAAGAGCGCAATTTCCCAGCTGACACTGTTCGTT1602              TCTTTGCTTCCCCGCGTTCCGCAGGCCGTAAGATTGAATTC1643                                 (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1643 nucleotides                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: AJ3463                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: mat peptide                                                     (B) LOCATION: 217..1482                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TCGCGAAGTAGCACCTGTCACTTTTGTCTCAAATATTAAATCGAATATCAATATACGGTC60                TGTTTATTGGAACGCATCCCAGTGGCTGAGACGCATCCGCTAAAGCCCCAGGAACCCTGT120               GCAGAAAGAAAACACTCCTCTGGCTAGGTAGACACAGTTTATAAAGGTAGAGTTGAGCGG180               GTAACTGTCAGCACGTAGATCGAAAGGTGCACAAAGGTGGCCCTGGTCGTACAG234                     MetAlaLeuValValGln                                                            15                                                                            AAATATGGCGGTTCCTCGCTTGAGAGTGCGGAACGCATTAGAAACGTC282                           LysTyrGlyGlySerSerLeuGluSerAlaGluArgIleArgAsnVal                              101520                                                                        GCTGAACGGATCGTTGCCACCAAGAAGGCTGGAAATGATGTCGTGGTT330                           AlaGluArgIleValAlaThrLysLysAlaGlyAsnAspValValVal                              253035                                                                        GTCTGCTCCGCAATGGGAGACACCACGGATGAACTTCTAGAACTTGCA378                           ValCysSerAlaMetGlyAspThrThrAspGluLeuLeuGluLeuAla                              404550                                                                        GCGGCAGTGAATCCCGTTCCGCCAGCTCGTGAAATGGATATGCTCCTG426                           AlaAlaValAsnProValProProAlaArgGluMetAspMetLeuLeu                              55606570                                                                      ACTGCTGGTGAGCGTATTTCTAACGCTCTCGTCGCCATGGCTATTGAG474                           ThrAlaGlyGluArgIleSerAsnAlaLeuValAlaMetAlaIleGlu                              758085                                                                        TCCCTTGGCGCAGAAGCTCAATCTTTCACTGGCTCTCAGGCTGGTGTG522                           SerLeuGlyAlaGluAlaGlnSerPheThrGlySerGlnAlaGlyVal                              9095100                                                                       CTCACCACCGAGCGCCACGGAAACGCACGCATTGTTGACGTCACACCG570                           LeuThrThrGluArgHisGlyAsnAlaArgIleValAspValThrPro                              105110115                                                                     GGTCGTGTGCGTGAAGCACTCGATGAGGGCAAGATCTGCATTGTTGCT618                           GlyArgValArgGluAlaLeuAspGluGlyLysIleCysIleValAla                              120125130                                                                     GGTTTTCAGGGTGTTAATAAAGAAACCCGCGATGTCACCACGTTGGGT666                           GlyPheGlnGlyValAsnLysGluThrArgAspValThrThrLeuGly                              135140145150                                                                  CGTGGTGGTTCTGACACCACTGCAGTTGCGTTGGCAGCTGCTTTGAAC714                           ArgGlyGlySerAspThrThrAlaValAlaLeuAlaAlaAlaLeuAsn                              155160165                                                                     GCTGATGTGTGTGAGATTTACTCGGACGTTGACGGTGTGTATACCGCT762                           AlaAspValCysGluIleTyrSerAspValAspGlyValTyrThrAla                              170175180                                                                     GACCCGCGCATCGTTCCTAATGCACAGAAGCTGGAAAAGCTCAGCTTC810                           AspProArgIleValProAsnAlaGlnLysLeuGluLysLeuSerPhe                              185190195                                                                     GAAGAAATGCTGGAACTTGCTGCTGTTGGCTCCAAGATTTTGGTGCTG858                           GluGluMetLeuGluLeuAlaAlaValGlySerLysIleLeuValLeu                              200205210                                                                     CGCAGTGTTGAATACGCTCGTGCATTCAATGTGCCACTTCGCGTACGC906                           ArgSerValGluTyrAlaArgAlaPheAsnValProLeuArgValArg                              215220225230                                                                  TCGTCTTATAGTAATGATCCCGGCACTTTGATTGCCGGCTCTATGGAG954                           SerSerTyrSerAsnAspProGlyThrLeuIleAlaGlySerMetGlu                              235240245                                                                     GATATTCCTGTGGAAGAAGCAGTCCTTACCGGTGTCGCAACCGACAAG1002                          AspIleProValGluGluAlaValLeuThrGlyValAlaThrAspLys                              250255260                                                                     TCCGAAGCCAAAGTAACCGTTCTGGGTATTTCCGATAAGCCAGGCGAG1050                          SerGluAlaLysValThrValLeuGlyIleSerAspLysProGlyGlu                              265270275                                                                     ACTGCCAAGGTTTTCCGTGCGTTGGCTGATGCAGAAATCAACATTGAC1098                          ThrAlaLysValPheArgAlaLeuAlaAspAlaGluIleAsnIleAsp                              280285290                                                                     ATGGTTCTGCAGAACGTCTCCTCTGTGGAAGACGGCACCACCGACATC1146                          MetValLeuGlnAsnValSerSerValGluAspGlyThrThrAspIle                              295300305310                                                                  ACGTTCACCTGCCCTCGCGCTGACGGACGCCGTGCGATGGAGATCTTG1194                          ThrPheThrCysProArgAlaAspGlyArgArgAlaMetGluIleLeu                              315320325                                                                     AAGAAGCTTCAGGTTCAGGGCAACTGGACCAATGTGCTTTACGACGAC1242                          LysLysLeuGlnValGlnGlyAsnTrpThrAsnValLeuTyrAspAsp                              330335340                                                                     CAGGTCGGCAAAGTCTCCCTCGTGGGTGCTGGCATGAAGTCTCACCCA1290                          GlnValGlyLysValSerLeuValGlyAlaGlyMetLysSerHisPro                              345350355                                                                     GGTGTTACCGCAGAGTTCATGGAAGCTCTGCGCGATGTCAACGTGAAC1338                          GlyValThrAlaGluPheMetGluAlaLeuArgAspValAsnValAsn                              360365370                                                                     ATCGAATTGATTTCCACCTCTGAGATCCGCATTTCCGTGCTGATCCGT1386                          IleGluLeuIleSerThrSerGluIleArgIleSerValLeuIleArg                              375380385390                                                                  GAAGATGATCTGGATGCTGCTGCACGTGCATTGCATGAGCAGTTCCAG1434                          GluAspAspLeuAspAlaAlaAlaArgAlaLeuHisGluGlnPheGln                              395400405                                                                     CTGGGCGGCGAAGACGAAGCCGTCGTTTATGCAGGCACCGGACGCTAA1482                          LeuGlyGlyGluAspGluAlaValValTyrAlaGlyThrGlyArg                                 410415420421                                                                  AGTTTTAAAGGAGTAGTTTTACAATGACCACCATCGCAGTTGTTGGTGCAACCGGCCAGG1542              TCGGCCAGGTTATGCGCACCCTTTTGGAAGAGCGCAATTTCCCAGCTGACACTGTTCGTT1602              TCTTTGCTTCCCCGCGTTCCGCAGGCCGTAAGATTGAATTC1643                                 (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1643 nucleotides                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: ATCC13869                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat peptide                                                     (B) LOCATION: 964..1482                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCGCGAAGTAGCACCTGTCACTTTTGTCTCAAATATTAAATCGAATATCAATATACGGTC60                TGTTTATTGGAACGCATCCCAGTGGCTGAGACGCATCCGCTAAAGCCCCAGGAACCCTGT120               GCAGAAAGAAAACACTCCTCTGGCTAGGTAGACACAGTTTATAAAGGTAGAGTTGAGCGG180               GTAACTGTCAGCACGTAGATCGAAAGGTGCACAAAGGTGGCCCTGGTCGTACAGAAATAT240               GGCGGTTCCTCGCTTGAGAGTGCGGAACGCATTAGAAACGTCGCTGAACGGATCGTTGCC300               ACCAAGAAGGCTGGAAATGATGTCGTGGTTGTCTGCTCCGCAATGGGAGACACCACGGAT360               GAACTTCTAGAACTTGCAGCGGCAGTGAATCCCGTTCCGCCAGCTCGTGAAATGGATATG420               CTCCTGACTGCTGGTGAGCGTATTTCTAACGCTCTCGTCGCCATGGCTATTGAGTCCCTT480               GGCGCAGAAGCTCAATCTTTCACTGGCTCTCAGGCTGGTGTGCTCACCACCGAGCGCCAC540               GGAAACGCACGCATTGTTGACGTCACACCGGGTCGTGTGCGTGAAGCACTCGATGAGGGC600               AAGATCTGCATTGTTGCTGGTTTTCAGGGTGTTAATAAAGAAACCCGCGATGTCACCACG660               TTGGGTCGTGGTGGTTCTGACACCACTGCAGTTGCGTTGGCAGCTGCTTTGAACGCTGAT720               GTGTGTGAGATTTACTCGGACGTTGACGGTGTGTATACCGCTGACCCGCGCATCGTTCCT780               AATGCACAGAAGCTGGAAAAGCTCAGCTTCGAAGAAATGCTGGAACTTGCTGCTGTTGGC840               TCCAAGATTTTGGTGCTGCGCAGTGTTGAATACGCTCGTGCATTCAATGTGCCACTTCGC900               GTACGCTCGTCTTATAGTAATGATCCCGGCACTTTGATTGCCGGCTCTATGGAGGATATT960               CCTGTGGAAGAAGCAGTCCTTACCGGTGTCGCAACCGACAAGTCCGAA1008                          MetGluGluAlaValLeuThrGlyValAlaThrAspLysSerGlu                                 151015                                                                        GCCAAAGTAACCGTTCTGGGTATTTCCGATAAGCCAGGCGAGGCTGCC1056                          AlaLysValThrValLeuGlyIleSerAspLysProGlyGluAlaAla                              202530                                                                        AAGGTTTTCCGTGCGTTGGCTGATGCAGAAATCAACATTGACATGGTT1104                          LysValPheArgAlaLeuAlaAspAlaGluIleAsnIleAspMetVal                              354045                                                                        CTGCAGAACGTCTCCTCTGTGGAAGACGGCACCACCGACATCACGTTC1152                          LeuGlnAsnValSerSerValGluAspGlyThrThrAspIleThrPhe                              505560                                                                        ACCTGCCCTCGCGCTGACGGACGCCGTGCGATGGAGATCTTGAAGAAG1200                          ThrCysProArgAlaAspGlyArgArgAlaMetGluIleLeuLysLys                              657075                                                                        CTTCAGGTTCAGGGCAACTGGACCAATGTGCTTTACGACGACCAGGTC1248                          LeuGlnValGlnGlyAsnTrpThrAsnValLeuTyrAspAspGlnVal                              80859095                                                                      GGCAAAGTCTCCCTCGTGGGTGCTGGCATGAAGTCTCACCCAGGTGTT1296                          GlyLysValSerLeuValGlyAlaGlyMetLysSerHisProGlyVal                              100105110                                                                     ACCGCAGAGTTCATGGAAGCTCTGCGCGATGTCAACGTGAACATCGAA1344                          ThrAlaGluPheMetGluAlaLeuArgAspValAsnValAsnIleGlu                              115120125                                                                     TTGATTTCCACCTCTGAGATCCGCATTTCCGTGCTGATCCGTGAAGAT1392                          LeuIleSerThrSerGluIleArgIleSerValLeuIleArgGluAsp                              130135140                                                                     GATCTGGATGCTGCTGCACGTGCATTGCATGAGCAGTTCCAGCTGGGC1440                          AspLeuAspAlaAlaAlaArgAlaLeuHisGluGlnPheGlnLeuGly                              145150155                                                                     GGCGAAGACGAAGCCGTCGTTTATGCAGGCACCGGACGCTAAAGTTTTAA1490                        GlyGluAspGluAlaValValTyrAlaGlyThrGlyArg                                       160165170172                                                                  AGGAGTAGTTTTACAATGACCACCATCGCAGTTGTTGGTGCAACCGGCCAGGTCGGCCAG1550              GTTATGCGCACCCTTTTGGAAGAGCGCAATTTCCCAGCTGACACTGTTCGTTTCTTTGCT1610              TCCCCGCGTTCCGCAGGCCGTAAGATTGAATTC1643                                         (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1643 nucleotides                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: AJ3463                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: mat peptide                                                     (B) LOCATION: 964..1482                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TCGCGAAGTAGCACCTGTCACTTTTGTCTCAAATATTAAATCGAATATCAATATACGGTC60                TGTTTATTGGAACGCATCCCAGTGGCTGAGACGCATCCGCTAAAGCCCCAGGAACCCTGT120               GCAGAAAGAAAACACTCCTCTGGCTAGGTAGACACAGTTTATAAAGGTAGAGTTGAGCGG180               GTAACTGTCAGCACGTAGATCGAAAGGTGCACAAAGGTGGCCCTGGTCGTACAGAAATAT240               GGCGGTTCCTCGCTTGAGAGTGCGGAACGCATTAGAAACGTCGCTGAACGGATCGTTGCC300               ACCAAGAAGGCTGGAAATGATGTCGTGGTTGTCTGCTCCGCAATGGGAGACACCACGGAT360               GAACTTCTAGAACTTGCAGCGGCAGTGAATCCCGTTCCGCCAGCTCGTGAAATGGATATG420               CTCCTGACTGCTGGTGAGCGTATTTCTAACGCTCTCGTCGCCATGGCTATTGAGTCCCTT480               GGCGCAGAAGCTCAATCTTTCACTGGCTCTCAGGCTGGTGTGCTCACCACCGAGCGCCAC540               GGAAACGCACGCATTGTTGACGTCACACCGGGTCGTGTGCGTGAAGCACTCGATGAGGGC600               AAGATCTGCATTGTTGCTGGTTTTCAGGGTGTTAATAAAGAAACCCGCGATGTCACCACG660               TTGGGTCGTGGTGGTTCTGACACCACTGCAGTTGCGTTGGCAGCTGCTTTGAACGCTGAT720               GTGTGTGAGATTTACTCGGACGTTGACGGTGTGTATACCGCTGACCCGCGCATCGTTCCT780               AATGCACAGAAGCTGGAAAAGCTCAGCTTCGAAGAAATGCTGGAACTTGCTGCTGTTGGC840               TCCAAGATTTTGGTGCTGCGCAGTGTTGAATACGCTCGTGCATTCAATGTGCCACTTCGC900               GTACGCTCGTCTTATAGTAATGATCCCGGCACTTTGATTGCCGGCTCTATGGAGGATATT960               CCTGTGGAAGAAGCAGTCCTTACCGGTGTCGCAACCGACAAGTCCGAA1008                          ValGluGluAlaValLeuThrGlyValAlaThrAspLysSerGlu                                 151015                                                                        GCCAAAGTAACCGTTCTGGGTATTTCCGATAAGCCAGGCGAGACTGCC1056                          AlaLysValThrValLeuGlyIleSerAspLysProGlyGluThrAla                              202530                                                                        AAGGTTTTCCGTGCGTTGGCTGATGCAGAAATCAACATTGACATGGTT1104                          LysValPheArgAlaLeuAlaAspAlaGluIleAsnIleAspMetVal                              354045                                                                        CTGCAGAACGTCTCCTCTGTGGAAGACGGCACCACCGACATCACGTTC1152                          LeuGlnAsnValSerSerValGluAspGlyThrThrAspIleThrPhe                              505560                                                                        ACCTGCCCTCGCGCTGACGGACGCCGTGCGATGGAGATCTTGAAGAAG1200                          ThrCysProArgAlaAspGlyArgArgAlaMetGluIleLeuLysLys                              657075                                                                        CTTCAGGTTCAGGGCAACTGGACCAATGTGCTTTACGACGACCAGGTC1248                          LeuGlnValGlnGlyAsnTrpThrAsnValLeuTyrAspAspGlnVal                              80859095                                                                      GGCAAAGTCTCCCTCGTGGGTGCTGGCATGAAGTCTCACCCAGGTGTT1296                          GlyLysValSerLeuValGlyAlaGlyMetLysSerHisProGlyVal                              100105110                                                                     ACCGCAGAGTTCATGGAAGCTCTGCGCGATGTCAACGTGAACATCGAA1344                          ThrAlaGluPheMetGluAlaLeuArgAspValAsnValAsnIleGlu                              115120125                                                                     TTGATTTCCACCTCTGAGATCCGCATTTCCGTGCTGATCCGTGAAGAT1392                          LeuIleSerThrSerGluIleArgIleSerValLeuIleArgGluAsp                              130135140                                                                     GATCTGGATGCTGCTGCACGTGCATTGCATGAGCAGTTCCAGCTGGGC1440                          AspLeuAspAlaAlaAlaArgAlaLeuHisGluGlnPheGlnLeuGly                              145150155                                                                     GGCGAAGACGAAGCCGTCGTTTATGCAGGCACCGGACGCTAAAGTTTTAA1490                        GlyGluAspGluAlaValValTyrAlaGlyThrGlyArg                                       160165170172                                                                  AGGAGTAGTTTTACAATGACCACCATCGCAGTTGTTGGTGCAACCGGCCAGGTCGGCCAG1550              GTTATGCGCACCCTTTTGGAAGAGCGCAATTTCCCAGCTGACACTGTTCGTTTCTTTGCT1610              TCCCCGCGTTCCGCAGGCCGTAAGATTGAATTC1643                                         (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1263 nucleotides                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: ATCC13869                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GTGGCCCTGGTCGTACAGAAATATGGCGGTTCCTCGCTTGAGAGTGCGGAACGCATTAGA60                AACGTCGCTGAACGGATCGTTGCCACCAAGAAGGCTGGAAATGATGTCGTGGTTGTCTGC120               TCCGCAATGGGAGACACCACGGATGAACTTCTAGAACTTGCAGCGGCAGTGAATCCCGTT180               CCGCCAGCTCGTGAAATGGATATGCTCCTGACTGCTGGTGAGCGTATTTCTAACGCTCTC240               GTCGCCATGGCTATTGAGTCCCTTGGCGCAGAAGCTCAATCTTTCACTGGCTCTCAGGCT300               GGTGTGCTCACCACCGAGCGCCACGGAAACGCACGCATTGTTGACGTCACACCGGGTCGT360               GTGCGTGAAGCACTCGATGAGGGCAAGATCTGCATTGTTGCTGGTTTTCAGGGTGTTAAT420               AAAGAAACCCGCGATGTCACCACGTTGGGTCGTGGTGGTTCTGACACCACTGCAGTTGCG480               TTGGCAGCTGCTTTGAACGCTGATGTGTGTGAGATTTACTCGGACGTTGACGGTGTGTAT540               ACCGCTGACCCGCGCATCGTTCCTAATGCACAGAAGCTGGAAAAGCTCAGCTTCGAAGAA600               ATGCTGGAACTTGCTGCTGTTGGCTCCAAGATTTTGGTGCTGCGCAGTGTTGAATACGCT660               CGTGCATTCAATGTGCCACTTCGCGTACGCTCGTCTTATAGTAATGATCCCGGCACTTTG720               ATTGCCGGCTCTATGGAGGATATTCCTGTGGAAGAAGCAGTCCTTACCGGTGTCGCAACC780               GACAAGTCCGAAGCCAAAGTAACCGTTCTGGGTATTTCCGATAAGCCAGGCGAGGCTGCC840               AAGGTTTTCCGTGCGTTGGCTGATGCAGAAATCAACATTGACATGGTTCTGCAGAACGTC900               TCCTCTGTGGAAGACGGCACCACCGACATCACGTTCACCTGCCCTCGCGCTGACGGACGC960               CGTGCGATGGAGATCTTGAAGAAGCTTCAGGTTCAGGGCAACTGGACCAATGTGCTTTAC1020              GACGACCAGGTCGGCAAAGTCTCCCTCGTGGGTGCTGGCATGAAGTCTCACCCAGGTGTT1080              ACCGCAGAGTTCATGGAAGCTCTGCGCGATGTCAACGTGAACATCGAATTGATTTCCACC1140              TCTGAGATCCGCATTTCCGTGCTGATCCGTGAAGATGATCTGGATGCTGCTGCACGTGCA1200              TTGCATGAGCAGTTCCAGCTGGGCGGCGAAGACGAAGCCGTCGTTTATGCAGGCACCGGA1260              CGC1263                                                                       (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1263 nucleotides                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: AJ3463                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GTGGCCCTGGTCGTACAGAAATATGGCGGTTCCTCGCTTGAGAGTGCGGAACGCATTAGA60                AACGTCGCTGAACGGATCGTTGCCACCAAGAAGGCTGGAAATGATGTCGTGGTTGTCTGC120               TCCGCAATGGGAGACACCACGGATGAACTTCTAGAACTTGCAGCGGCAGTGAATCCCGTT180               CCGCCAGCTCGTGAAATGGATATGCTCCTGACTGCTGGTGAGCGTATTTCTAACGCTCTC240               GTCGCCATGGCTATTGAGTCCCTTGGCGCAGAAGCTCAATCTTTCACTGGCTCTCAGGCT300               GGTGTGCTCACCACCGAGCGCCACGGAAACGCACGCATTGTTGACGTCACACCGGGTCGT360               GTGCGTGAAGCACTCGATGAGGGCAAGATCTGCATTGTTGCTGGTTTTCAGGGTGTTAAT420               AAAGAAACCCGCGATGTCACCACGTTGGGTCGTGGTGGTTCTGACACCACTGCAGTTGCG480               TTGGCAGCTGCTTTGAACGCTGATGTGTGTGAGATTTACTCGGACGTTGACGGTGTGTAT540               ACCGCTGACCCGCGCATCGTTCCTAATGCACAGAAGCTGGAAAAGCTCAGCTTCGAAGAA600               ATGCTGGAACTTGCTGCTGTTGGCTCCAAGATTTTGGTGCTGCGCAGTGTTGAATACGCT660               CGTGCATTCAATGTGCCACTTCGCGTACGCTCGTCTTATAGTAATGATCCCGGCACTTTG720               ATTGCCGGCTCTATGGAGGATATTCCTGTGGAAGAAGCAGTCCTTACCGGTGTCGCAACC780               GACAAGTCCGAAGCCAAAGTAACCGTTCTGGGTATTTCCGATAAGCCAGGCGAGACTGCC840               AAGGTTTTCCGTGCGTTGGCTGATGCAGAAATCAACATTGACATGGTTCTGCAGAACGTC900               TCCTCTGTGGAAGACGGCACCACCGACATCACGTTCACCTGCCCTCGCGCTGACGGACGC960               CGTGCGATGGAGATCTTGAAGAAGCTTCAGGTTCAGGGCAACTGGACCAATGTGCTTTAC1020              GACGACCAGGTCGGCAAAGTCTCCCTCGTGGGTGCTGGCATGAAGTCTCACCCAGGTGTT1080              ACCGCAGAGTTCATGGAAGCTCTGCGCGATGTCAACGTGAACATCGAATTGATTTCCACC1140              TCTGAGATCCGCATTTCCGTGCTGATCCGTGAAGATGATCTGGATGCTGCTGCACGTGCA1200              TTGCATGAGCAGTTCCAGCTGGGCGGCGAAGACGAAGCCGTCGTTTATGCAGGCACCGGA1260              CGC1263                                                                       (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 516 nucleotides                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: ATCC13869                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GTGGAAGAAGCAGTCCTTACCGGTGTCGCAACCGACAAGTCCGAAGCCAAAGTAACCGTT60                CTGGGTATTTCCGATAAGCCAGGCGAGGCTGCCAAGGTTTTCCGTGCGTTGGCTGATGCA120               GAAATCAACATTGACATGGTTCTGCAGAACGTCTCCTCTGTGGAAGACGGCACCACCGAC180               ATCACGTTCACCTGCCCTCGCGCTGACGGACGCCGTGCGATGGAGATCTTGAAGAAGCTT240               CAGGTTCAGGGCAACTGGACCAATGTGCTTTACGACGACCAGGTCGGCAAAGTCTCCCTC300               GTGGGTGCTGGCATGAAGTCTCACCCAGGTGTTACCGCAGAGTTCATGGAAGCTCTGCGC360               GATGTCAACGTGAACATCGAATTGATTTCCACCTCTGAGATCCGCATTTCCGTGCTGATC420               CGTGAAGATGATCTGGATGCTGCTGCACGTGCATTGCATGAGCAGTTCCAGCTGGGCGGC480               GAAGACGAAGCCGTCGTTTATGCAGGCACCGGACGC516                                       (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 516 nucleotides                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Corynebacterium glutamicum                                      (B) STRAIN: AJ3463                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GTGGAAGAAGCAGTCCTTACCGGTGTCGCAACCGACAAGTCCGAAGCCAAAGTAACCGTT60                CTGGGTATTTCCGATAAGCCAGGCGAGACTGCCAAGGTTTTCCGTGCGTTGGCTGATGCA120               GAAATCAACATTGACATGGTTCTGCAGAACGTCTCCTCTGTGGAAGACGGCACCACCGAC180               ATCACGTTCACCTGCCCTCGCGCTGACGGACGCCGTGCGATGGAGATCTTGAAGAAGCTT240               CAGGTTCAGGGCAACTGGACCAATGTGCTTTACGACGACCAGGTCGGCAAAGTCTCCCTC300               GTGGGTGCTGGCATGAAGTCTCACCCAGGTGTTACCGCAGAGTTCATGGAAGCTCTGCGC360               GATGTCAACGTGAACATCGAATTGATTTCCACCTCTGAGATCCGCATTTCCGTGCTGATC420               CGTGAAGATGATCTGGATGCTGCTGCACGTGCATTGCATGAGCAGTTCCAGCTGGGCGGC480               GAAGACGAAGCCGTCGTTTATGCAGGCACCGGACGC516                                       (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TCGCGAAGTAGCACCTGTCACTT23                                                     (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ACGGAATTCAATCTTACGGCC21                                                       (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GCCAGGCGAGCGTGCCAAGGTTT23                                                     (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GCCAGGCGAGGATGCCAAGGTTT23                                                     (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GCCAGGCGAGTGTGCCAAGGTTT23                                                     (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GCCAGGCGAGTTTGCCAAGGTTT23                                                     (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GCCAGGCGAGCCTGCCAAGGTTT23                                                     (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GCCAGGCGAGTCTGCCAAGGTTT23                                                     (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GCCAGGCGAGTATGCCAAGGTTT23                                                     (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 nucleotides                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GCCAGGCGAGGTTGCCAAGGTTT23                                                     __________________________________________________________________________

What is claimed is:
 1. A recombinant DNA fragment coding for anaspartokinase α-subunit protein originating from a bacterium belongingto the genus Corynebacterium, in which synergistic feedback inhibitionby L-lysine and L-threonine is substantially desensitized, said proteinhaving an amino acid sequence defined by SEQ ID NO: 4, or a sequence inwhich the 279th residue in said amino acid sequence is changed to anamino acid other than Ala and other than acidic amino acids.
 2. Arecombinant DNA fragment coding for an aspartokinase β-subunit proteinoriginating from a bacterium belonging to the genus Corynebacterium, inwhich synergistic feedback inhibition by L-lysine and L-threonine issubstantially desensitized, said protein having an amino acid sequencedefined by SEQ ID NO: 6, or a sequence in which a 30th residue in saidamino acid sequence is changed to an amino acid residue other than Alaand other than acidic amino acids.
 3. The DNA fragment according toclaim 1, having a nucleotide sequence defined by SEQ ID NO:
 12. 4. TheDNA fragment according to claim 2, having a nucleotide sequence definedby SEQ ID NO:
 14. 5. Recombinant DNA containing the DNA fragment asdefined in claim 1 and being replicable in microorganisms belonging tothe genus Corynebacterium.
 6. A transformant obtained by introducing therecombinant DNA as defined in claim 5 into a microorganism belonging tothe genus Corynebacterium, wherein the specific activity ofaspartokinase is increased 2-20 times as compared with a parent strain,and synergistic feedback inhibition by L-lysine and L-threonine orfeedback inhibition by L-lysine alone exerted on the activity ofaspartokinase is substantially desensitized.
 7. A method of producingL-lysine, comprising the steps of:cultivating the transformant asdefined in claim 6 in an appropriate medium; and separating producedL-lysine.
 8. An aspartokinase α-subunit protein originating from abacterium belonging to the genus Corynebacterium, in which synergisticfeedback inhibition by L-lysine and L-threonine is substantiallydesensitized, said protein having an amino acid sequence defined by SEQID NO: 4, or a sequence in which a 270th residue in said amino acidsequence is changed to an amino acid residue other than Ala and otherthan acidic amino acids.
 9. An aspartokinase β-subunit proteinoriginating from a bacterium belonging to the genus Corynebacterium, inwhich synergistic feedback inhibition by L-lysine and L-threonine issubstantially desensitized, said protein having an amino acid sequencedefined by SEQ ID NO: 6, or a sequence in which a 30th residue in saidamino acid sequence is changed to an amino acid residue other than Alaand other than acidic amino acids.
 10. Recombinant DNA containing theDNA fragment as defined in claim 2 and being replicable inmicroorganisms belonging to the genus Corynebacterium.
 11. RecombinantDNA containing the DNA fragment as defined in claim 3 and beingreplicable in microorganisms belonging to the genus Corynebacterium. 12.Recombinant DNA containing the DNA fragment as defined in claim 4 andbeing replicable in microorganisms belonging to the genusCorynebacterium.